Halo everybody. I am doing immunocytochemistry staining of pluripotent stem cell-derived neurons seeded on coverslips. During ICC procedure, I fixed the cells using 4% PFA for 15 minutes and then washed the coverslips using D-PBS with Ca2+ and Mg2+. Unfortunately, many of them were floating in the solution and I can't use the coverslips for subsequent staining processes. does anybody has some suggestion for that problem ? FYI that the PSCs were seeded on matrigel coated 24 well plate and seeded with the initial density culture approximately 28000 cells
There are two things I have done that may help.
After the neurons are plated, they are overlaid with additional matrigel once a week during maturation.
Fixation is done in two steps. Only half the media is removed, and that is replaced with an equal volume of 4% PFA. After 15 minutes this is removed and replaced with 4% PFA.
You can find many ways of keeping your cells attached to the coverslips, one of them is coating your coverslips with poly-L-Lysine. Another could be coating with type I or IV collagen that may also keep your cells attached.
Another suggestion I could give you is to be extremely careful when washing your samples. I mean, when you have to add your 4% PFA and then wash it, try to suck and release the volumes keeping your pipet tip the closest possible to the well wall. Also try to be as gentle as possible, this way you will lower the possibility to create a strong flux that would eventually make your cells detach from the coverslip.
It is better to use SuperFrost slides. Though rather expensive, they are the best for attachment of PFA fixed material. If the neurones still float, try taking the excess liquid with a tiny capillary, monitoring the process under the dissecting microscope. and add other solutions very gently
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